Multicomponent assemblies having enhanced binding properties for diagnosis and therapy

ABSTRACT

An organized mobile multicomponent conjugate (OMMC) and method of using to enhance binding of weakly binding compounds to a target. A lamellar structure containing at least two binding compounds is assembled under conditions in which the binding compounds self-regulate in or on the lamellar structure, forming a cooperative ensemble that is capable of binding with enhanced affinity to a complementary affinity site on a target. Each binding compound is bound to the lamellar surface, and may be connected by a linker. The OMMC may contain an effector molecule, such as a diagnostic or therapeutic agent, for administration to a patient who is then diagnosed or treated using the effector molecule.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of pending U.S. patent application Ser.Nos. 12/603,438 and 12/603,402, both of which were filed on 23 Oct.2009, and both of which are divisions of U.S. Pat. No. 7,713,514 filedon 4 Aug. 2006, which is a division of U.S. Pat. No. 7,087,212 filed on17 Aug. 2001, all of which are entitled “Multicomponent AssembliesHaving Enhanced Binding Properties for Diagnosis and Therapy”, and allof which are incorporated by reference in their entireties.

FIELD OF THE INVENTION

This invention pertains to methods and compositions for enhancedtargeted binding to selectively deliver diagnostic and therapeuticagents to target tissues and organs.

BACKGROUND OF THE INVENTION

Non-covalent intermolecular forces (e.g., electrostatic, hydrogenbonding and van der Waals interactions) play a vital role in manybiological processes. Examples of such processes include enzymecatalysis, drug-receptor interaction, antigen-antibody interaction,biotin-avidin interaction, DNA double helix formation, phagocytosis,pigmentation in plants and animals, and cellular transport.

Targeted delivery of medically useful molecules is well known and hasbeen successfully applied in diagnostic and therapeutic applications. Inconventional bioconjugate chemistry, almost all of the conjugates areprepared by covalent attachment of various effector molecules, such asdrugs, dyes, hormones, magnetic resonance imaging agents, echogenicagents, radiopharmaceuticals, or chemotherapeutic agents, to bioactivecarriers, such as antibodies, peptides and peptidomimetics,carbohydrates, or small molecule receptor agonists and antagonists. Forexample, in diagnostic medicine, various radionuclide and radionuclidechelates covalently attached to antibodies, hormones, peptides,peptidomimetics, and the like have been used to detect lesions such astumors, inflammation, ischemia, and thrombi (Tenenbaum et al.,Radiolabeled somatostatin analog scintigraphy in differentiated thyroidcarcinoma, J. Nucl. Med., 1995, 36, 807-810; Haldemann et al.,Somatostatin receptor scintigraphy in central nervous system tumors:Role of blood-brain barrier permeability. J. Nucl. Med., 1995, 36,403-410; Babich et al., Technetium-99m-labeled chemotactic peptides:Comparison with indium-111-labeled white blood cells for localizingacute bacterial infection in the rabbit. J. Nucl. Med., 1993, 34,2176-2181; Knight et al., Thrombus imaging with technetium-99m-labeledsynthetic peptides based upon the binding domain of a monoclonalantibody to activated platelets. J. Nucl. Med., 1994, 35, 282-288).Thus, the target site may be cells such as tumor cells, platelets,erythrocytes, leukocytes, macrophages, vascular endothelial cells,myocardial cells, hepatocytes, etc., or the extracellular matrixsurrounding these cells.

In addition to the direct administration of biologically activecompounds into the body, molecules such as these have also beenencapsulated within organized amphiphilic aggregates such as a liposome,vesicle, or other multilamellar structures. The aggregates are thendelivered to the particular organs or tissues of interest (U.S. Pat.Nos. 5,985,852; 5,785,969; and 5,542,935).

A requirement for targeted delivery is a strong interaction between thebinding compound or targeting moiety (e.g., ligand) and the target orsite of attachment (e.g., receptor) in the formation of a complex (e.g.,ligand receptor complex). The dissociation constant (K_(d)) value of thecomplex should typically be in the nanomolar range. Compounds exhibitingK_(d) values from about 100 nM and upward are considered weak bindingcompounds and are not generally considered to be useful for targetedimaging and therapeutic applications. However, there are many in vivobiological processes that do operate using multiple weak-bindinginteractions. These include, for example, enzyme-substrate complexes andcell adhesion molecules, which operate in the micromolar binding range.Cell adhesion molecules such as E, P, and L selectins are importantbiological modulators implicated in inflammatory and thrombolyticprocesses. (McEver, Selectin-carbohydrate interactions duringinflammation and metastasis, Glycoconj. J., 1997, 14(5), 585-91; McEveret al., Leukocyte trafficking mediated by selectin-carbohydrateinteractions, J. Biol. Chem., 1995, 270(19), 11025-8; Bischoff, Celladhesion and angiogenesis. J. Clin. Invest., 1997, 100 (11 Suppl),S37-39; Lesley et al., CD44 in inflammation and metastasis, Glycoconj.J., 1997, 14(5), 611-22; Siegelman et al., Activation and interaction ofCD44 and hyaluronin in immunological systems. J. Leukoc. Biol., 1999,66(2), 315-21).

Previous work in this area involved the use of only high-binding ligands(Torchilin et al., Preservation of antimyosin antibody activity aftercovalent coupling to liposomes, Biochem. Biophys. Res. Commun., 1979,89(4), 1114-9; Allen et al., A new strategy for attachment of antibodiesto sterically stabilized liposomes resulting in efficient targeting tocancer cells. Biochim. Biophys. Acta, 1995, 1237, 99-108; Zalipsky etal., Peptide attachment to extremities of liposomal surface grafted PEGchains: preparation of the long-circulating form of lamininpentapeptide, YIGSR. Bioconjug. Chem., 1995, 6(6), 705-8). Althoughincreased binding was observed in this system, a cooperative effect wasnot needed because the ligand was already endowed with sufficientaffinity for targeting purposes. In contrast, weakly binding ligandspresented a formidable challenge.

There is considerable effort to improve the binding affinity ofrelatively weakly binding selectins and selectin mimics, and to attachthem covalently to effector molecules for imaging and therapeuticpurposes (Fukuda et al., Peptide mimic of E-selectin ligand inhibitssialyl Lewis X-dependent lung colonization of tumor cells, CancerResearch, 2000, 60, 450-456). However, such efforts usually involvesynthesizing and screening large numbers of new chemical entities todiscover the ones that exhibit substantially improved bindingproperties. Furthermore, simple conjugation of effector moieties to analready weakly binding carrier is expected to result in a bioconjugatewhose bioactivity is either greatly diminished or obviated altogether.Thus, there is a need for a simple method to enhance the affinity of anyweakly binding targeted molecules in order to enhance their usefulnessfor diagnostic and/or therapeutic purposes.

SUMMARY OF THE INVENTION

The invention is directed to compositions and methods that enhancebinding of a compound, particularly a weakly binding compound, to itstarget site in a patient.

An organized mobile multifunctional conjugate (OMMC) assembly isprepared and provided to a patient. The OMMC assembly is prepared byanchoring at least two binding compounds to a lamellar structure. In oneembodiment, one binding compound is an anionic compound, and anotherbinding compound is a saccharide, and the lamellar structure is aliposome, microsphere, micelle, etc. The binding compounds incorporatedinto the lamellar structure are mobile, and self-adjust relative to eachother to form an OMMC ensemble. The OMMC ensemble binds to a target thatcontains at least two complementary affinity sites for the bindingcompounds. An agent, also called an effector molecule, may be attachedto and/or contained within the lamellar structure to provide a targeteddiagnostic and/or therapeutic agent to a patient to whom the OMMC isadministered.

These and other advantages of the inventive compounds and methods willbe apparent in light of the following figures, description, andexamples.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic illustration of the general structure of anorganized mobile multifunctional conjugate (OMMC) assembly.

FIG. 2 is a schematic illustration of the steps in forming and bindingan OMMC ensemble to a target.

FIG. 3 is a schematic two-dimensional illustration of an OMMC ensemblebinding to a target.

FIG. 4 shows results of composite OMMC ensemble binding to humanendothelial cells.

DETAILED DESCRIPTION

In non-covalent interactions, although the free energy of associationper unit interaction is quite small (less than about 40 kJ/interaction),the cumulative effect of multiple points of interaction along twosurfaces can be substantial. This can lead to strong specific bindingbetween the binding entities. Enhancement of binding due to thecooperative effect of multiple binding domains between two interactingunits is well known in biological chemistry. For example, themultivalence of most antigens leads to a synergistic effect, in whichthe binding of two antigen molecules by antibody is greater than the sumof the individual antigen-antibody links (J. Kuby, Immunology, 2^(nd)Edition, 1994, W.H. Freeman & Co., New York, pp. 138-139).

In the invention, enhanced binding of a binding compound that bindsweakly (that is, a K_(d) of about 100 nM or higher) to a target isachieved using a multicomponent assembly. Specifically, an organizedmobile multifunctional conjugate (OMMC) assembly is prepared byanchoring at least two binding compounds to a lamellar structure. In oneembodiment, the binding compounds are an anionic compound and asaccharide, and the lamellar structure is a liposome, microsphere,micelle, etc. The OMMC assembly is provided to a patient. The bindingcompounds incorporated into the lamellar structure are mobile, andself-adjust relative to each other to form an OMMC ensemble, which bindsto a target that contains at least two complementary affinity sites forthe binding compounds.

“Organized” refers to assemblies which are capable of assuming close toan optimum configuration on an assembly surface for binding atparticular sites of interaction. “Mobile” refers to the degree offreedom within the assembly to allow complementary binding compounds orcomponents to move independently within the lamellar structure withrespect to each other to assume the optimum configuration.“Multifunctional conjugate” refers to two or more amphiphilic componentsthat each contain a binding compound for a target complementary affinitysite. The binding compounds of the ensemble cooperatively bind to thetarget with enhanced binding affinity, without the specific bindingregions being contained within a single molecule. Furthermore, bindingcompounds in the inventive ensemble, constructed so that they expressover a volume of space occupied outside of the ensemble, move withinthis space to interrogate the space until they find their respectivebinding complements.

An agent or effector molecule, for example, a contrast agent, may beattached to and/or contained within the lamellar structure to providetargeted delivery of the agent in a patient to whom the OMMC assembly isadministered Enhanced binding of these binding compounds may beexploited for diagnostic and therapeutic purposes.

The invention overcomes problems presented by weakly binding compoundsby using novel OMMC assemblies. These dynamic self-adjusting OMMCassemblies allow increased targeting specificity and affinity of thebinding compound to affinity sites in the target, particularly incomparison with an aggregate of individual active binding components.

The invention is also related to using the described OMMC assemblies fordelivering effector molecules to the target tissues or organs fordiagnostic and/or therapeutic purposes. Appropriate OMMC assemblies thatcontain a diagnostic and/or therapeutic agent or effector moleculeattached to, incorporated within, or contained within the lamellarstructure are prepared. Two or more binding compounds are incorporatedon or into the surface of the OMMC assemblies. The OMMC assemblies areadministered in an effective amount to a patient, and the bindingcompounds move about in or on the surface and self-adjust their relativepositions and configurations in or on the assembly to bind to affinitysites at a target. A diagnostic or therapeutic procedure is thereafterperformed on the patient. The procedure may use an imaging technique,for example, X-ray, computed tomography (CT), positron emissiontomography (PET), magnetic resonance (MR), ultrasound, optics orradiography. The procedure may also be an in vitro diagnostic procedure,or a chemo-, photo-, or radiotherapeutic procedure, using methods knownin the art.

One advantage of the present invention is that it does not requirehigh-binding molecules to achieve a cooperative effect.

The general structure of an OMMC assembly 10 is shown in FIG. 1. Alamellar structure 13, the outer surface forming a type of surroundingshell 11, contains binding compounds 16 a, 16 b. The binding compounds16 a, 16 b may be a ligand, an antibody, a peptide, a peptidomimetic, acarbohydrate, or a small molecule receptor agonist or antagonist ligand.In the lamellar structure 13 are regions 12 a, 12 b that incorporate thebinding compounds 16 a, 16 b on or in the assembly 10. The bindingcompounds 16 a, 16 b contain binding regions or epitopes 14 a, 14 b.These epitopes 14 a, 14 b may individually have weak binding and bind toa target only through a cooperative effect. Flexible linkers 18 a, 18 bconnect or tether the binding regions 14 a, 14 b to the anchoringregions 12 a, 12 b.

Anchor regions 12 a, 12 b are hydrophobic and self-assemble to minimizeits aspect relative to the aqueous phase. Anchor regions 12 a, 12 b maybe the same or different, and may be one or more of CH₃(CH₂)_(a)—W,CF₃(CH₂)_(a)—W, CF₃(CF₂)_(a)—W, CF₃(CF₂)_(a)CH₂CH₂—W,CH₃(CH₂)_(a)—O—(CH₂)_(b)—W, CF₃(CF₂)_(a)—O—(CH₂)_(b)—W,CH₃(CH₂)_(a)—S—(CH₂)_(b)—W, or CH₃(CH₂)_(a)—S—S—(CH₂)_(b)—W, andR¹O₂CCH₂(CHW)CO₂R¹, wherein a and b range from 16 to 32, W is aconnector unit of —O—, —CO—, —CO₂—, —OCO—, O₂CO—, —S—, —SO—, —SO₂—,—OSO—, —OSO₂—, —NH—, —NHCO—, —NHCS, —NHSO₂—, —PO₂—, —OPO₂—, —PO₂H—, or—OPO₂H—, and R is a normal alkyl radical containing 16 to 24 carbonatoms. Other hydrophobic material may also be contained within theanchor regions 12 a, 12 b.

Binding regions 14 a, 14 b are hydrophilic and are solvated with anaqueous phase. Binding regions 14 a, 14 b may be the same or different,and may be amino acids, peptides, and peptidomimics; mono-, di-, andoligosaccharides, such as C or O monosaccharides and glycosides, C or Oglucosides, glucose, galactose, fucose, glycomimics, and glycopeptides;sialic acid, carminic acid, or anionic compounds of —(CH₂)d—CO₂ ⁻,—(CH₂)_(d)—SO₃ ⁻, —(CH₂)_(d)—OSO₃ ⁻, —(CH₂)_(d—PO) ₃ ^(═),—(CH₂)_(d)—OPO₃ ^(═), —Ar—SO₃ ⁻, DTPA, EDTA, DOTA, or EGTA, where dranges from 1 to 10.

Linkers 18 a, 18 b may be the same or different, and may bepolysorbates, polyglycerols, polypeptides, polynucleotides,polysaccharides, polyvinylpyrrolidones, polyvinylalcohols,polyethyleneglycols such as polyethythene glycols having a molecularweight in the range of 1,000 to 4,000, polyglycolate, polylactate, orcopolymers derived from any of the above groups.

The lamellar structure 13 may be CH₃—(CH₂)_(e)—X, CH₃—(CF₂)_(e)—X,CF₃—(CF₂)_(e)—X, CF₃—(CF₂)_(f)—O—(CH₂)_(g)—X,CH₃—(CH₂)_(f)—S—(CH₂)_(g)—X, CH₃—(CH₂)_(f)—S—S—(CH₂)_(g)—X,CH₃—(CH₂)_(g)CO₂(CH₂)_(h)—X, CH₃—(CH₂)_(f)CONH(CH₂)_(g)—X,CH₃—(CH₂)_(f)NHCONH(CH₂)_(g)—X, CH₃—(CH₂)_(f)OCONH(CH₂)_(g)—X,CH₃—(CH₂)_(f)NH(CH₂)_(g)—X, CH₃—(CH₂)_(f)N[(CH₂)_(g)]₂—X,CH₃—(CH₂)_(f)SO(CH₂)_(g)—X, CH₃—(CH₂)_(f)SO₂(CH₂)_(g)—X,CH₃—(CH₂)_(m)NH(CH₂)_(f)CO₂(CH₂)_(g)—X,CH₃—[(CH₂)_(f)]₂N(CH₂)_(g)CONH(CH₂)_(h)—X, R²O₂CCH₂(CHY)CO₂R², and/orR²O₂CCH₂CH₂(CHY)CO₂R²; where R² is a normal alkyl radical containing 16to 24 carbon atoms, e ranges from 16 to 32, f, g, and h range from 1 to15, X is carboxylate, sulfonate, sulfate, phosphate, or phosphonate, Yis —(CH₂)_(k)—X, —OCO(CH₂)_(k)—X, —NHCO(CH₂)_(k)—X, or —CH₂OCO(CH₂)₂—CO₂⁻, and k is 2 to 6. The lamellar structure 13 may also be salts ofdocosanoic, tetracosanoic, hexacosanoic, octacosanoic, and triacontanoicacids.

The various chemical compositions of the anchor regions, bindingregions, linkers, and lamellar structure are as listed in the followingtables.

TABLE 1 Anchors A1 and/or A2 (12a or 12b) CH₃(CH₂)_(a)—W CF₃(CH₂)_(a)—WCF₃(CF₂)_(a)—W CF₃(CF₂)_(a)CH₂CH₂—W CH₃(CH₂)_(a)—O—(CH₂)_(b)—WCF₃(CF₂)_(a)—O—(CH₂)_(b)—W CH₃(CH₂)_(a)—S—(CH₂)_(b)—WCH₃(CH₂)_(a)—S—S—(CH₂)_(b)—W a, b = 16-32 R¹O₂CCH₂(CHW)CO₂R¹ R¹ is anormal alkyl radical containing 16-24 carbon atoms. W —O— —CO— —CO₂——O₂C— —OCO— —O₂CO— —S— —SO— —OSO— —OSO₂— —SO₂— —OPO₂H— —NH— —NHCO——NHCS— —NHSO₂— —PO₂H —PO₂— —OPO₂—

TABLE 2 Binding Region (Epitopes) B1 and/or B2 (14a, 14b) amino acidspeptides (1-20 amino acids) peptidomimics monosaccharidesoligosaccharides (1-10) glycomimics glycopeptides anionic compounds suchas: —(CH₂)_(d)—CO₂ ⁻ —(CH₂)_(d)—SO₃ ⁻ —(CH₂)_(d)—OSO₃ ⁻ —(CH₂)_(d)—PO₃ ⁼—(CH₂)_(d)—OPO₃ ⁼ d = 1-10 —Ar—SO₃ ⁻ DTPA EDTA DOTA EGTA C— or O—monosaccharides and glycosides such as: glucose mannose fucose galactoseglucosamine mannosamine galactosamine sialic acid flavonoidsisoflavonones C— or O— glucosides such as, but not limited to: rutinneohesperidin dihydrochalone phloridizin hesperidin hesperidin methylchalcone naringenin esculin carminic acid family including, but notlimited to, carmine, 18b-glycyrrhetinic acid and salt B1 B2oligosaccharide derived from the glycan —O(CH₂)_(1or2)CO₂ ⁻ family ofcarbohydrate including, but not limited to: hyaluronic acid—O(CH₂)_(1or2)SO₃ ⁻ heparin —O(CH₂)_(1or2)SO₄ ⁻ chondroitin sulfate—O(CH₂)_(1or2)PO₄ ⁻ dermatan mono or disaccharide including, but notlimited to: galactose fucose glucose mannose hyaluronic acid

TABLE 3 Lamellar Structure CH₃—(CH₂)_(e)—X CH₃—(CF₂)_(e)—XCF₃—(CF₂)_(e)—X CF₃—(CF₂)_(f)—O—(CH₂)_(g)—X CH₃—(CH₂)_(f)—S—(CH₂)_(g)—XCH₃—(CH₂)_(f)—S—S—(CH₂)_(g)—X CH₃—(CH₂)_(g)CO₂(CH₂)_(h)—XCH₃—(CH₂)_(f)CONH(CH₂)_(g)—X CH₃—(CH₂)_(f)NHCONH(CH₂)_(g)—XCH₃—(CH₂)_(f)OCONH(CH₂)_(g)—X CH₃—(CH₂)_(f)NH(CH₂)_(g)—XCH₃—(CH₂)_(f)N[(CH₂)_(g)]₂—X CH₃—(CH₂)_(f)SO(CH₂)_(g)—XCH₃—(CH₂)_(f)SO₂(CH₂)_(g)—X CH₃—(CH₂)_(m)NH(CH₂)_(f)CO₂(CH₂)_(g)—XCH₃—[(CH₂)_(f)]₂N(CH₂)_(g)CONH(CH₂)_(h)—X e = 16-32 f, g, h = 1-15 X =carboxylate, sulfonate, sulfate, phosphate, phosphonate salts of:docosanoic acid tetracosanoic acid hexacosanoic acid octacosanoic acidtriacontanoic acid R²O₂CCH₂(CHY)CO₂R² R²O₂CCH₂CH₂(CHY)CO₂R² R² is anormal alkyl radical containing 16-24 carbon atoms Y = —(CH₂)_(k)—X,—NHCO(CH₂)_(k)—X, —OCO(CH₂)_(k)—X, —CH₂OCO(CH₂)₂—CO₂ ⁻, k = 1-6 X =carboxylate, sulfate, phosphate

TABLE 4 Linkers L1 and/or L2 (18a, 18b) polysorbates polyglycerolspolypeptides polynucleotides polysaccharides polyvinylpyrrolidonespolyvinylalcohols polyethyleneglycols polyglycolate polylactatepoly(ethyleneglycol)_(p) (p = 40-150)

In one embodiment of the invention, OMMC assemblies 10 have the generalstructure shown in FIG. 1 where anchors 12 a, 12 b may be the same ordifferent and are selected from the group or groups consisting ofCH₃(CH₂)_(a)—W, wherein a ranges from 16 to 24 and W is a connector unitselected from the group consisting of —O—, —CO—, —CO₂—, —OCO—, —S—,—SO—, —SO₂—, —OSO₂—, —NH—, —NHCO—, —NHCS—, —NHSO₂—, —PO₂H—, or —OPO₂H—.Binding regions 14 a, 14 b may be the same or different and are selectedfrom the group consisting of C or O monosaccharides and glycosides, witholigosaccharides containing 1 to 10 furanose or pyranose units, aminoacids, or peptides containing 1 to 20 amino acid residues, flavonoidsand Isoflavonones, C— or O— glucosides (rutin, neohesperidindihydrochalone, phloridizin, hesperidin, hesperidin methyl chalcone,naringenin, esculin); carminic acid family members including, but notlimited to, carmine, 18b-glycyrrhetinic acid and salts. Linkers 18 a, 18b may be the same or different and are selected from the groupconsisting of polyethyleneglycols, polyglycolate, and polylactate.Lamellar structure 13 is selected from the group consisting ofCH₃—(CH₂)_(e)—X, wherein e ranges from 16 to 32, and X is selected fromthe group consisting of carboxylate, sulfonate, sulfate, phosphate, andphosphonate.

In another embodiment of the invention, OMMC assemblies 10 have thegeneral structure as shown in FIG. 1, where binding region 14 a is anoligosaccharide derived from the glycan family of carbohydrates thatincludes, but is not limited to, hyaluronic acid, heparin, chondroitinsulfate, and dermatan, or a mono- or disaccharide including, but notlimited to, galactose, fucose, glucose, mannose, or hyaluronic acid. Inthis embodiment, binding region 14 b is selected from the groupconsisting of —O(CH₂)_(1 or 2)CO₂ ⁻; —O(CH₂)_(1 or 2)SO₃ ⁻;—O(CH₂)_(1 or 2)SO₄ ⁻; —O(CH₂)_(1 or 2)PO₄ ⁻. Linkers 18 a, 18 b areselected from the group consisting of poly(ethyleneglycol)_(p) with pbetween 40 and 150 units. Lamellar structure 13 is selected from thegroup consisting of salts of docosanoic, tetracosanoic, hexacosanoic,octacosanoic and triacontanoic acid.

Enhancement of the affinity of weakly binding compounds 16 a, 16 b isaccomplished according to the following method, as schematicallyillustrated in FIGS. 2 and 3. Two or more binding compounds 16 a, 16 b,such as weakly binding compounds, are added to the lamellar structure 13to form an OMMC assembly 10 a. The binding compounds 16 a, 16 b areinitially randomly distributed throughout the lamellar structure 13.

The binding compounds 16 a, 16 b on assembly 10, in the presence of thecomplementary affinity sites 15 a, 15 b on the target 17, move about thelamellar structure in or on the surface and self-adjust their relativepositions and configurations by differential movement to produce anoptimized ensemble structure 30, bound to complementary affinity sites15 a, 15 b, such as receptors. The mobility of the binding compounds 16a, 16 b in and on the OMMC assembly 10 a is similar to the differentialmotion of amphiphilic molecules within a cell membrane surface. Optimumbinding occurs by an approach to equilibrium process. All components ofthe OMMC assembly 10 have a dynamic motion, both up and down and side toside. In addition, linkers 18 a, 18 b act as tethers and can also“float” within a given volume of space over the surface of the assembly10 in the aqueous phase. If an expressed region of a target 17 havingmany affinity sites 15 a, 15 b is encountered, multiple bindings startto occur and a dynamic equilibrium is established: as one set of bindingcompounds 16 a, 16 b detach, another set of binding compounds 16 c, 16 d(not shown) adjacent to binding compounds 16 a, 16 b are within aspatial distance to attach. At a point, the number of attachments to atarget 17 is sufficient to halt motion of the assembly 10, essential“locking” it onto the target 17. The binding compounds (e.g., ligands)16 a, 16 b in the optimized assembly 10 then bind via their bindingregions 14 a, 14 b, in a cooperative fashion, to their respectiveepitopes in receptors 15 a, 15 b of target 17. Thus, the binding domains14 a, 14 b, which individually may have weak interaction with a giventarget surface, form a self-optimizing composition, resulting in greatlyincreased binding and targeting effectiveness.

As previously stated, the OMMC assembly 10 can accommodate variouseffector molecules 30. Effector molecules 30 include, for example,fluids (e.g., a gas or liquid fluorocarbon or superheated fluid) usefulfor ultrasonic procedures, fluorescent compounds useful for biomedicaloptical procedures, paramagnetic agents useful for magnetic resonanceimaging procedures, radionuclides for nuclear medicine applications suchas I-123, I-131, Tc-99m, Re-186, Re-188, SM-152, Ho-155, Bi-202, andLu-177, and X-ray opacification agents for X-ray or computed tomographyprocedures. Examples of each of these effector molecules and theireffective doses and uses are well known to those of skill in the art asdescribed in, for example, Fritzsch et al., Eur. Radiol. 2, 2-13 (1992);Rubaltelli, Photodiagnostic and Phototherapeutic Techniques in Medicine,in Documento Editoriale, G. Joni and C. Perria (Eds.), Milano, 1995,101-107; Andreoni et al., Tumour Photosensitization by ChemotherapeuticDrugs, Biologia, 1993, No. 3, 43-6; Knoefel, I.E.P.T. Section 76, Vol.1, Pergamon Press, Oxford and New York, 1971, Chapter 2: Organic IodineCompounds as X-ray Contrast Media; Rumack et al., Diagnostic Ultrasound,Vol. 1, Mosby Year Book, St. Louis, Chapter 3, Contrast Agents inDiagnostic Ultrasound, 30-42 (1992); Forsberg et al., Ultrasonics 1998,Clinical Applications of Ultrasound Contrast Agents, February,36(1-5):695-701; Goldberg et al., Ultrasound Med. Biol. 1994, 20(4):319-33, Ultrasound Contrast Agents: A Review; deJong and Ten Cate,Ultrasonics 1996 June, 34(2-5):587-90, New Ultrasound Contrast Agentsand Technological Innovations; Goldberg, Clin. Diag. Ultrasound 1993,28: 35-45, Ultrasound Contrast Agents; Dalla Palma and Bertolotto, Eur.Radiol., 1999, Suppl 3:S338-42, Introduction to Ultrasound ContrastAgents: Physics Overview; Ophir and Parker, Ultrasound Med. Biol. 1989,15(4):319-33, Contrast Agents in Diagnostic Ultrasound; Klibanov et al.,MAGMA 1999 August, 8(3):177-184, Targeting and Ultrasound Imaging ofMicrobubble-Based Contrast Agents; Makdisi and Versland, Targeted Diag.Ther. 1991, 4:151-162, Asialoglycoproteins as Radiodiagnostic Agents forDetection of Hepatic Masses and Evaluation of Liver Function; WO9004943; U.S. Pat. No. 5,312,617; Tilcock et al., Radiology 1989, 171:77-80, Liposomal Gd-DTPA: Preparation and Characterization ofRelaxivity, each of which is expressly incorporated by reference hereinin its entirety.

The dose of effector molecules depends on the type of imaging or therapybeing performed (nuclear, optical, magnetic resonance, ultrasound,optical acoustic or X-ray contrast). In general, the doses necessary forthese procedures vary. For examples, for nuclear imaging, depending onthe type of emission, only a few micrograms are necessary. Opticalimaging agents require tens to hundreds of milligrams for a human dose.Ultrasound imaging requires about 0.2 to 10×10⁹ microbubbles/mL, with atypical dose of around 1 mL (stabilized with a few milligrams ofcomposite) of the described formulations; in one embodiment 250 μL wasused. Magnetic resonance imaging typically requires 50 to 1000 milligramdoses in solution of metal chelates. X-ray contrast imaging requiresseveral grams of material in solution for opacification. In oneembodiment, targeted X-ray contrast imaging of the liver is performed,with the effector molecules taken up by the hepatic cells.

The effector molecule 30 can be attached, either directly or indirectly,to the assembly 10 or contained within the assembly 10. For example, anechogenic agent and/or MRI agent may be a fluid contained within thevoid of the assembly 10. A radionuclide, optical and/or cytotoxic agentmay be anywhere in the assembly 10 (in the void, or on binding compound16 a, 16 b). A chemotherapeutic agent in the assembly 10 can be releasedby ultrasonication to induce therapy, as described in U.S. Pat. Nos.5,770,222 and 5,733,572, and WO 684386, each of which is expresslyincorporated by reference herein in its entirety. A paramagnetic agentmay be in the assembly 10 at the region of the linkers 18 a, 18 b orepitopes 14 a, 14 b so that it is in contact with the aqueous milieu. Aneffector molecule 30 may be an echogenic agent such as a microbubblecontaining one or more fluids, a fluorophore or chromophore capable ofemitting or absorbing electromagnetic radiation in the range of about300-1200 nm, a radioactive molecule capable of emitting alpha, beta, orgamma radiation, a paramagnetic agent, X-ray opacification agent, and/orchemotherapeutic agent. The effector molecule may be a paramagneticagent selected from Gd-DTPA, Gd-DOTA, Gd-DTPA-bis (methoxyethyl) amide,and Mn-EDTA.

In one embodiment, agent 30 is an echogenic agent, fluorophore orchromophore capable of absorbing or emitting light in the range of 300to 1200 nm, a radiopharmaceutical agent such as diethylenetriaminepentaacetic acid (DTPA) or1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetraacetic acid (DOTA),chelates of beta and gamma emitting isotopes such as Lu, Sm, In, Ru, Tc;cytotoxic agents including, but not limited to, fluorouracil,fluorouridine, etc., and/or sulfa antibiotics including, but not limitedto N′-(w-thiazolyl)sulfanilamide, sulfmethoxazole, sulfisoxazole, andsulfisomidine.

In another embodiment, agent 30 is an echogenic water insolubleperfluoropropane or perfluorobutane gas, or is one or more of sulfurhexafluoride, tetrafluoromethane, hexafluoroethane, octafluoropropane,decafluorobutane, dodecafluoropentane, and perfluorohexane. Theechogenic agents include mixtures of these gases with a common gas, ormixture of gases such as nitrogen, oxygen, helium, neon, argon, xenon,and carbon dioxide. Superheated fluids or azeotropes of fluorocarbonsand perfluorocarbons may be used. Agent 30 may be an optical tracer,such as fluoresceins, rhodamines, cyanines, indocyanines (e.g.,indocyanine green), squaraines, phenothiazines, porphyrins, and azocompounds.

In yet another embodiment, the agent 30 may be a bioactive molecule,such as E, P, and L selectins, selectin mimetics, hyaluronic acid,heparin, CD44, ICAM 1, and the like. In this case, the affinity bindingsites 15 a, 15 b in the target 17 generally belong to the class ofcarbohydrate receptors which are found, for example, on vascularendothelial cells, leukocytes, or platelets.

The structure 13 of the OMMC assembly 10 may be constructed by methodsknown to one skilled in the art including, but not limited to, micelles,liposomes, emulsions, microparticles, microbubbles, vesicles bounded byuni- or multi-lamellar membranes, nano- and micro-particles, nano- andmicro-aggregates, nano- and micro-envelopes, and living cells. In oneembodiment, a unilamellar structure surrounds a fluid-filled core, suchas a gas/liquid emulsion (bubble). In another embodiment, the lamellarstructure 13 is a hybrid containing some amount of waxy substance orpolymer that is blended with binding compounds 16 a, 16 b to form theanchoring regions 12 a, 12 b. Methods of formation include, but are notlimited to, use of an ultrasound microfluidizer, use of a colloid mill,ultramixing, and the like as described in U.S. Pat. Nos. 5,510,118;5,543,133; and 5,552,133, each of which is expressly incorporated byreference herein in its entirety.

The size of the assembly 10 can be varied according to the type ofapplication contemplated. The size of the assembly 10 may, in oneembodiment, be less than 5 microns. In another embodiment, the size ofthe assembly may range from 1 nm to 10⁴ nm, and can be controlled byknown methods, as described in U.S. Pat. No. 6,001,335, which isexpressly incorporated by reference herein in its entirety.

The inventive compositions have broad clinical utility. Uses include,but are not limited to, diagnostic imaging of tumors, of inflammation(both noninfectious and infectious), and of impaired vasculature; laserguided endoscopic examination of lesion sites; diagnostic imaging usingultrasound, magnetic resonance, radiographic and optical imagingprocedures, and photodynamic therapy and chemotherapy of tumors andinfection. Other potential but not limiting uses for targeted specificbinding are vascular imaging of the endothelium, imaging ofneo-vascularized and inflamed tissue, imaging changes in the circulationin or around necrotic tissue, detection of metastases due to theheterogeneous expression of target molecules, and alleviation ofangiogenesis inflammation after re-vascularization of infarcted tissueafter stenosis removal.

The OMMC assembly 10 can be formulated into diagnostic or therapeuticcompositions for enteral, parenteral, or oral administration. Thesephysiologically acceptable compositions contain an effective amount ofan OMMC assembly 10, along with conventional physiological carriersand/or excipients appropriate for the type of administrationcontemplated. As one example, parenteral administration mayadvantageously contain a sterile aqueous solution or suspension ofassemblies 10 having a concentration in the range of about 0.1 μM toabout 10 mM (from about 10⁶ to about 10¹⁰ particles/mL). Parenteralformulations may have an assembly 10 concentration from about 100 μM toabout 2 mM. Such solutions also may contain pharmaceutically acceptablebuffers, emulsifiers, surfactants, and, optionally, electrolytes such assodium chloride. As another example, enteral administration may varywidely, as is well known in the art. In general, such formulations areliquids which include an effective amount of the OMMC assembly 10 in anaqueous solution or suspension. Enteral composition may optionallyinclude buffers, surfactants, emulsifiers, thixotropic agents, and thelike. Compositions for oral administration may also contain flavoringagents and other ingredients for enhancing their organoleptic qualities.

The compositions are administered in doses effective to achieve thedesired diagnostic and/or therapeutic objective. Such doses may varywidely, depending upon the particular OMMC assembly 10 employed, theorgans or tissues to be examined, the equipment employed in the clinicalprocedure, and the like. The type of equipment needed may also varywidely, depending on the type of imaging modality contemplated.

The following examples serve to illustrate some aspects of theinvention.

EXAMPLE 1

Preparation of OMMC Assembly Having One Binding Domain and a TerminalCarboxylate Binding Region, and Containing Gas (Docosanoate (C₂₁CO₂—);Octacosanoate (C₂₇CO₂—); and Succinylated PEG[100]stearate Formulatedwith n-Perfluorobutane).

To an 8 dram (29.6 mL) vial was added docosanoic acid (10.0 mg, Sigma,˜99%); octacosanoic acid (10 mg, Aldrich, ˜98%) and 22 mg ofsuccinylated PEG[100]stearate weighed on a Mettler® analytical balance.The mixture was dissolved into about 1 mL of dichloromethane (distilledMallinckrodt Ultimar®) using mild heating. Dichloromethane wascompletely removed by the application of heat under a flow of nitrogen(Air Products, High Purity Grade). Excess sodium hydrogen carbonate(about 100 mg, Aldrich, ˜99.7%) was placed in the flask along with 10 mLof 0.9% sodium chloride solution used for irrigation (Baxter, USP).

The tip of the insonation probe, Heat Systems Sonicator® UltrasonicProcessor XL (Heat Systems, Inc., Farmingdale, N.Y.) Model XL 2020, in asound box was positioned, using a small lab jack, midway into the liquidof the vial. An intensity level of 5 was applied for 2 minutes and 30seconds. The sample, at about 70° C., was taken out of the sound box soas to cool the vial contents under a flow of tap water to a temperaturebelow about 50° C. as measured by a FLUKE® 52 K/J thermometer fittedwith a K thermocouple. The tip of the insonation horn was repositionedjust under the surface of the liquid. A gas flow (30 to 50 mL/min) wasestablished from a lecture bottle into the bottom of the vial usingmicro-tubing (PVDF or equivalent) at a power setting of 5 for 45seconds, and then was increased to a power setting of 7 for 10 seconds.The vial containing a thick white suspension of gas microenvelopes wascapped and allowed to cool in a room temperature water bath.

After centrifugation at 1,000×g (2,000 rpm) for 1 minute, the liquidphase (bottom), containing excess matrix material, was completelyreplaced with a decafluorobutane-saturated 0.9% sodium chloridesolution. A resuspension procedure followed by centrifugation wasrepeated two additional times. A 30 mL plastic syringe barrel fittedwith a two-way valve was filled with decafluorobutane (97%, Pfalz &Bauer, Inc.). The sample was suspended thoroughly and poured into thesyringe barrel. A stopper was fitted onto the top of the syringe barrel.The sample was allowed to stand for ten minutes. The lower 5 mL wasdrained into a 10 mL syringe vial filled with decafluorobutane andsealed. A sample was taken for Coulter® analysis and was refrigerated asmuch as possible.

EXAMPLE 2

Preparation of OMMC assembly with One Binding Domain and a TerminalGalactosamide Binding Region, and Containing Gas (Docosanoate (C₂₁CO₂—);Octacosanoate (C₂₇CO₂—); and Octahexadecanoyl[poly(ethylene glycol)5,000]Succinamic-Galactosyl Amide Formulated with n-Perfluorobutane).

To an 8 dram (29.6 mL) vial was added docosanoic acid (10.0 mg, Sigma,˜99%); octacosanoic acid (10 mg, Aldrich, ˜98%) and 22 mg ofoctahexadecanoyl[poly(ethylene glycol) 5,000]succinamicgalactosyl amide(prepared by the reaction of succinylated PEG[100]stearate, DCC andD-galactosamine (Aldrich)) weighed on a Mettler® analytical balance. Themixture was dissolved into about 1 mL dichloromethane (distilledMallinckrodt Ultimar®) using mild heating. The dichloromethane wascompletely removed by the application of heat under a flow of nitrogen(Air Products, High Purity Grade). Excess sodium hydrogen carbonate(˜100 mg, Aldrich®, ˜99.7%) was placed in the flask along with 10 mL of0.9% sodium chloride solution used for irrigation (Baxter, USP).

The tip of the insonation probe, Heat Systems Sonicator® UltrasonicProcessor XL (Heat Systems, Inc., Farmingdale, N.Y.) Model XL 2020, in asound box was positioned, using a small lab jack, midway into the liquidof the vial. The intensity level of 5 was applied for 2 minutes and 30seconds. The sample, at about 70° C., was taken out of the sound box soas to cool the vial contents under a flow of tap water to a temperaturebelow about 50° C. as measured by a FLUKE® 52 K/J thermometer fittedwith a K thermocouple. The tip of the insonation horn was repositionedjust under the surface of the liquid. A gas flow (30 to 50 mL/min) wasestablished from a lecture bottle into the bottom of the vial usingmicro-tubing (PVDF or equivalent) at a power setting of 5 for 45seconds, and then was increased to a power setting of 7 for 10 seconds.The vial of thick white suspension of gas microenvelopes was capped andallowed to cool in a room temperature water bath.

After centrifugation at 1,000×g (2,000 rpm) for 1 minute, the liquidphase (bottom) containing excess matrix material was completely replacedwith a decafluorobutane-saturated 0.9% sodium chloride solution. Aresuspension procedure followed by centrifugation was repeated twoadditional times. A 30 cc plastic syringe barrel fitted with a two-wayvalve was filled with decafluorobutane (97%, Pfalz & Bauer, Inc.). Thesample was suspended thoroughly and poured into the syringe barrel. Astopper was fitted onto the top of the syringe barrel. The sample wasallowed to stand for ten minutes. The lower 5 mL was drained into a 10mL syringe vial filled with decafluorobutane and sealed. A sample wastaken for Coulter® analysis and was refrigerated as much as possible.

EXAMPLE 3

Preparation of OMMC Assembly with Two Binding Domains (TerminalCarboxylate, Terminal Galactosamide) and Containing Gas (Docosanoate(C₂₁CO₂—); Octacosanoate (C₂₇CO₂—); Succinylated PEG[100]stearate andOctahexadecanoyl[poly(ethylene glycol) 5,000]Succinamic-Galactosyl AmideFormulated with n-Perfluorobutane).

To an 8 dram (29.6 mL) vial was added docosanoic acid (10.0 mg, Sigma,˜99%); octacosanoic acid (10 mg, Aldrich, ˜98%), 11 mg ofoctahexadecanoyl[poly(ethylene glycol) 5,000]succinamicgalactosyl amide(prepared by the reaction of succinylated PEG[100]stearate, DCC andD-galactosamine (Aldrich)), and 11 mg of succinylated PEG[100]stearateweighed out on a Mettler® analytical balance. The mixture was dissolvedinto about 1 mL dichloromethane (distilled Mallinckrodt Ultimar®) usingmild heating. The dichloromethane was completely removed by theapplication of heat under a flow of nitrogen (Air Products, High PurityGrade). Excess sodium hydrogen carbonate (˜100 mg, Aldrich, ˜99.7%) wasplaced in the flask along with 10 mL of 0.9% sodium chloride solutionused for irrigation (Baxter, USP). The tip of the insonation probe, HeatSystems Sonicator® Ultrasonic Processor XL (Heat Systems, Inc.,Farmingdale, N.Y.) Model XL 2020, in a sound box was positioned, using asmall lab jack, midway into the liquid of the vial. The intensity levelof 5 was applied for 2 minutes and 30 seconds. The sample, at about 70°C., was taken out of the sound box so as to cool the vial contents undera flow of tap water to a temperature below about 50° C. as measured by aFLUKE® 52 K/J thermometer fitted with a K thermocouple.

The tip of the insonation horn was repositioned just under the surfaceof the liquid. A gas flow (30 to 50 mL/min) was established from alecture bottle into the bottom of the vial using micro-tubing (PVDF orequivalent) at a power setting of 5 for 45 seconds, and then increasedto a power setting of 7 for 10 seconds. The vial of thick whitesuspension of gas microenvelopes was capped and allowed to cool in aroom temperature water bath. The liquid phase (bottom) containing excessmatrix material was completely replaced with decafluorobutane saturated0.9% sodium chloride solution used for irrigation (Baxter, USP). The tipof the insonation probe, Heat Systems Sonicator® Ultrasonic Processor XL(Heat Systems, Inc., Farmingdale, N.Y.) Model XL 2020, in a sound boxwas positioned, using a small lab jack, midway into the liquid of thevial. The intensity level of 5 was applied for 2 minutes and 30 seconds.The sample at about 70° C. was taken out of the sound box so as to coolthe vial contents under a flow of tap water to a temperature below about50° C. as measured by a FLUKE® 52 K/J thermometer fitted with a Kthermocouple. The tip of the insonation horn was repositioned just underthe surface of the liquid. A gas flow (30 to 50 mL/min) was establishedfrom a lecture bottle into the bottom of the vial using micro-tubing(PVDF or equivalent) at a power setting of 5 for 45 seconds, and thenincreased to a power setting of 7 for 10 seconds. The vial of thickwhite suspension of gas microenvelopes was capped and allowed to cool ina room temperature water bath. The liquid phase (bottom) containingexcess matrix material was completely replaced with decafluorobutanesaturated 0.9% sodium chloride solution after centrifugation at 1,000×g(2,000 rpm) for 1 minute. Resuspension procedure followed bycentrifugation was repeated two additional times.

A 30 mL plastic syringe barrel fitted with a two-way valve was filledwith decafluorobutane (97%, Pfalz & Bauer, Inc.). The sample wassuspended thoroughly and poured into the syringe barrel. A stopper wasfitted onto the top of the syringe barrel. The sample was allowed tostand for ten minutes. The lower 5 mL was drained into a 10 mL syringevial filled with decafluorobutane and sealed. A sample was taken forCoulter® analysis, and was kept refrigerated as much as possible.

EXAMPLE 4 Binding of OMMC Assemblies

The assemblies prepared in Examples 1, 2, and 3 were incubated over a 30minute period with stirring in the presence of a semi-confluentmonolayer of human umbilical cord endothelial cells (HUVEC), (ATCCCRL-1730). The cells were placed into adjacent wells in the presence ofmedia with and without heparin present. Media was removed and the cellswere washed.

After the 30 minute incubation, the cells were examined microscopically(200×) with phase contrast for binding of the assemblies. Little bindingwas noted with assemblies containing only one binding domain of charged(sulfate anion alone) carboxylate (data not shown). Some bindingoccurred with assemblies containing surface N-acyl-mannoside or-galactoside terminated microenvelopes (data not shown). As shown inFIG. 4, binding was greatly increased, and there was excellent surfacecoverage of activated cells, with assemblies containing the combinationof sulfate and N-acyl-mannoside or N-acyl-galactoside compositemicroenvelopes.

In all cases, binding was substantially blocked by the presence ofheparin or hyaluronic acid oligosaccharides. This is likely due toheparin or hyaluronic acid oligosaccharide in free solution blocking thebinding of the assemblies by competing for complementary affinity siteson the cell membrane or extracellular matrix.

EXAMPLE 5

Preparation of Gas-Filled Microenvelopes Comprised of Docosanoate(C₂₁CO₂—); Octacosanoate (C₂₇CO₂—); and Octahexadecanoyl[poly(ethyleneglycol) 5,000]Succinamic-Galactosyl Amide and Sulfated PEG[100]stearateFormulated with n-Perfluorobutane.

To an 8 dram (29.6 mL) vial was added docosanoic acid (10.0 mg, Sigma,˜99%); octacosanoic acid (10 mg, Aldrich, ˜98%) and 10 mg each ofoctahexadecanoyl [poly(ethylene glycol) 5,000] succinamicgalactosylamide (prepared by the reaction of succinylated PEG[100]stearate, DCCand D-galactosamine (Aldrich)) and sulfated PEG[100]stearate (preparedby the reaction of PEG[100]stearate, DCC and sulfuric acid) weighed on aMettler® analytical balance. The mixture was dissolved into about 1 mLacetone (distilled Mallinckrodt Ultimar®) using mild heating. Theacetone was completely removed by the application of heat under a flowof nitrogen (Air Products, High Purity Grade). Excess sodium hydrogencarbonate (˜100 mg, Aldrich, ˜99.7%) was placed in the flask along with10 mL of 0.9% sodium chloride solution used for irrigation (Baxter,USP). The tip of the insonation probe, Heat Systems Sonicator®Ultrasonic Processor XL (Heat Systems, Inc., Farmingdale, N.Y.) Model XL2020, in a sound box was positioned, using a small lab jack, midway intothe liquid of the vial. An intensity level of 5 was applied for 2minutes and 30 seconds. The sample, at about 70° C., was taken out ofthe sound box so as to cool the vial contents under a flow of tap waterto a temperature below about 50° C. as measured by a FLUKE® 52 K/Jthermometer fitted with a K thermocouple. The tip of the insonation hornwas repositioned just under the surface of the liquid. A gas flow (30 to50 mL/min) was established from a lecture bottle into the bottom of thevial using micro-tubing (PVDF or equivalent) at a power setting of 5 for45 seconds, and then was increased to a power setting of 7 for 10seconds. The vial of thick white suspension of gas microenvelopes wascapped and allowed to cool in a room temperature water bath.

After centrifugation at 1,000×g (2,000 rpm) for 1 minute, the liquidphase (bottom) containing excess matrix material was completely replacedwith a decafluorobutane-saturated 0.9% sodium chloride solution. A 30 ccplastic syringe barrel fitted with a two-way valve was filled withdecafluorobutane (97%, Pfalz & Bauer, Inc.). The sample was suspendedthoroughly and poured into the syringe barrel. A stopper was fitted ontothe top of the syringe barrel. The sample was allowed to stand fortwenty minutes. The lower 5 mL was drained into a 10 mL syringe vialfilled with decafluorobutane and sealed. The sample was keptrefrigerated as much as possible.

EXAMPLE 6

Preparation of Gas-Filled Microenvelopes Comprised of Docosanoate(C₂₁CO₂—); Octacosanoate (C₂₇CO₂—); and Octahexadecanoyl[poly(ethyleneglycol) 5,000]Succinamic-Mannosyl Amide Formulated withn-Perfluorobutane.

To an 8 dram (29.6 mL) vial was added docosanoic acid (10.0 mg, Sigma,˜99%); octacosanoic acid (10 mg, Aldrich, ˜98%) and 20 mg ofoctahexadecanoyl[poly(ethylene glycol) 5,000]succinamicmannosyl amideweighed on a Mettler® analytical balance. The procedure as described inExample 5 was followed. The resultant sample was kept refrigerated asmuch as possible.

EXAMPLE 7

Preparation of Gas-Filled Microenvelopes Comprised of Docosanoate(C₂₁CO₂—); Octacosanoate (C₂₇CO₂—); and Octahexadecanoyl[poly(ethyleneglycol) 5,000]Succinamic-Mannosyl Amide and Sulfated PEG[100]stearateFormulated with n-Perfluorobutane.

To an 8 dram (29.6 mL) vial was added docosanoic acid (10.0 mg, Sigma,˜99%); octacosanoic acid (10 mg, Aldrich, ˜98%) and 10 mg each ofoctahexadecanoyl[poly(ethylene glycol) 5,000]succinamicmannosyl amide(prepared by the reaction of succinylated PEG[100]stearate, DCC andD-mannosamine (Aldrich)) and sulfated PEG[100]stearate (prepared by thereaction of PEG[100]stearate, DCC and sulfuric acid) weighed out on aMettler® analytical balance. The procedure as described in Example 5 wasfollowed. The resultant sample was kept refrigerated as much aspossible.

EXAMPLE 8

Preparation of Gas-Filled Microenvelopes Comprised of Docosanoate(C₂₁CO₂—); Octacosanoate (C₂₇CO₂—); and Sulfated PEG[100]stearateFormulated with n-Perfluorobutane.

To an 8 dram (29.6 mL) vial was added docosanoic acid (10.0 mg, Sigma,˜99%); octacosanoic acid (10 mg, Aldrich, ˜98%) and 20 mg of sulfatedPEG[100]stearate weighed on a Mettler® analytical balance. The procedureas described in Example 5 was followed. The resultant sample was keptrefrigerated as much as possible.

EXAMPLE 9 Binding of the Gas-Filled Microenvelopes to ActivatedEndothelial Cells (HUVEC)

The microenvelopes (bubbles) prepared in Examples 5, 6, 7, and 8 wereincubated over a 30-minute period with stirring in the presence of amonolayer of human endothelial cells (HUVEC, human umbilical cordendothelial cells, ATCC CRL-1730). The three samples generated asdescribed in Examples 5-8 were incubated in adjacent wells in thepresence of media with and without heparin present.

After removing the media, washing, and microscopic examination (400×)with phase contrast, little binding was noted with only charged (sulfateanion alone) microenvelopes. Some binding occurred with surface withN-acyl-mannoside or -galactoside terminated microenvelopes. Greatlyincreased, excellent surface coverage of bubbles binding to activatedcells was observed with the combination of sulfated and N-acyl-mannosideor -galactoside composite microenvelopes. The binding in all cases wascompletely blocked by the presence of heparin.

While the invention has been disclosed by reference to the details ofpreferred embodiments of the invention, it is to be understood that thedisclosure is intended in an illustrative rather than in a limitingsense, as it is contemplated that modifications will readily occur tothose skilled in the art, within the spirit of the invention and thescope of the appended claims.

1. A biocompatible organized mobile multicomponent conjugate (OMMC)comprising: (A) a lamellar structure comprisingCH₃—(CH₂)_(f)CONH(CH₂)_(g)—X, wherein: (1) X is selected fromcarboxylate, sulfonate, sulfate, phosphate, and phosphonate; and (2)each off and g is independently 1-15; and (B) a first binding compoundand a second binding compound, each of which is anchored to the lamellarstructure, the first binding compound capable of binding to an exogenousfirst affinity site, and the second binding compound capable of bindingto an exogenous second affinity site, wherein the first and secondbinding compounds are mobile and self-adjust relative to the lamellarstructure to allow for cooperative binding of the first and secondbinding compounds to said first and second affinity sites, respectively,wherein each binding compound independently comprises: (1) an anchorregion that anchors the binding compound to the lamellar structure, theanchor region being selected from CH₃(CH₂)_(a)—W,CH₃(CH₂)_(a)—O—(CH₂)_(b)—W, CH₃(CH₂)_(a)—S—(CH₂)_(b)—W,R¹O₂CCH₂(CHW)CO₂R¹, CF₃(CH₂)_(a)—W, CF₃(CF₂)_(a)—W,CF₃(CF₂)_(a)CH₂CH₂—W, CF₃(CF₂)_(a)—O—(CH₂)_(b)—W, andCH₃(CH₂)_(a)—S—S—(CH₂)_(b)—W, wherein: (a) each of a and b ranges from16 to 32, inclusive; (b) W is selected from —O—, —CO—, —CO₂—, —OCO—,—O₂CO—, —S—, —SO—, —SO₂—, —NH—, —NHCO—, —NHCS—, —NHSO₂—, —PO₂H—,—OPO₂H—, —PO₂—, and —OPO₂—; and (c) R¹ is an alkyl radical containing 16to 24 carbon atoms; and (2) a binding region selected from amino acids,peptides, peptidomimics, monosaccharides, disaccharides,oligosaccharides, sialic acid, carminic acid, and anionic compounds of—(CH₂)_(d)—CO₂ ⁻, —(CH₂)_(d)—SO₃ ⁻, —(CH₂)_(d)—OSO₃ ⁻, —(CH₂)_(d)—PO₃^(═), —(CH₂)_(d)—OPO₃ ^(═), —Ar—SO₃ ⁻, DTPA, EDTA, DOTA, and EGTA,wherein d ranges from 1 to 10, inclusive; and (3) a linker that connectsthe anchor region to the binding region, the linker being selected frompolysorbates, polyglycerols, polypeptides, polynucleotides,polysaccharides, polyvinylpyrrolidones, polyvinylalcohols,polyethyleneglycols, polyglycolate, polylactate, and copolymers of anyof the members of this group.
 2. The conjugate of claim 1, wherein X iscarboxylate.
 3. The conjugate of claim 1, wherein the anchor region ofthe first binding compound, the second binding compound, or each of thefirst and second binding compounds is CH₃(CH₂)_(a)—W.
 4. The conjugateof claim 3, wherein W is —O—.
 5. The conjugate of claim 3, wherein W is—CO—.
 6. The conjugate of claim 3, wherein W is —CO₂—.
 7. The conjugateof claim 3, wherein W is —NH—.
 8. The conjugate of claim 3, wherein W is—O₂CO—.
 9. The conjugate of claim 3, wherein W is —NHCO—.
 10. Theconjugate of claim 3, wherein W is —S—.
 11. The conjugate of claim 1,wherein the anchor region of the first binding compound, the secondbinding compound, or each of the first and second binding compounds isR¹O₂CCH₂(CHW)CO₂R¹.
 12. The conjugate of claim 11, wherein W is —O—. 13.The conjugate of claim 11, wherein W is —CO—.
 14. The conjugate of claim11, wherein W is —CO₂—.
 15. The conjugate of claim 11, wherein W is—NH—.
 16. The conjugate of claim 11, wherein W is —O₂CO—.
 17. Theconjugate of claim 11, wherein W is —NHCO—.
 18. The conjugate of claim11, wherein W is —S—.
 19. The conjugate of claim 1, wherein the anchorregion of the first binding compound, the second binding compound, oreach of the first and second binding compounds isCH₃(CH₂)_(a)—O—(CH₂)_(b)—W.
 20. The conjugate of claim 19, wherein W is—O—.
 21. The conjugate of claim 19, wherein W is —CO—.
 22. The conjugateof claim 19, wherein W is —CO₂—.
 23. The conjugate of claim 19, whereinW is —NH—.
 24. The conjugate of claim 19, wherein W is —O₂CO—.
 25. Theconjugate of claim 19, wherein W is —NHCO—.
 26. The conjugate of claim19, wherein W is —S—.
 27. The conjugate of claim 1, wherein the bindingregion of the first binding compound, the second binding compound, oreach of the first and second binding compounds is an amino acid.
 28. Theconjugate of claim 1, wherein the binding region of the first bindingcompound, the second binding compound, or each of the first and secondbinding compounds is a monosaccharide.
 29. The conjugate of claim 28,wherein the binding region of the first binding compound, the secondbinding compound, or each of the first and second binding compounds isselected from C monosaccharides and O monosaccharides.
 30. The conjugateof claim 1, wherein the binding region of the first binding compound,the second binding compound, or each of the first and second bindingcompounds is a disaccharide.
 31. The conjugate of claim 1, wherein thebinding region of the first binding compound, the second bindingcompound, or each of the first and second binding compounds is sialicacid.
 32. The conjugate of claim 1, wherein the binding region of thefirst binding compound, the second binding compound, or each of thefirst and second binding compounds is a glycoside of a monosaccharide orsialic acid.
 33. The conjugate of claim 1, wherein the binding region ofthe first binding compound, the second binding compound, or each of thefirst and second binding compounds is a monosaccharide selected frommannose, glucose, fucose, and galactose.
 34. The conjugate of claim 1,wherein the binding region of the first binding compound, the secondbinding compound, or each of the first and second binding compounds is amonosaccharide selected from glucosamine, mannosamine, andgalactosamine.
 35. The conjugate of claim 1, wherein the binding regionof the first binding compound, the second binding compound, or each ofthe first and second binding compounds is DTPA.
 36. The conjugate ofclaim 1, wherein the binding region of the first binding compound, thesecond binding compound, or each of the first and second bindingcompounds is DOTA.
 37. The conjugate of claim 1, wherein the bindingregion of the first binding compound, the second binding compound, oreach of the first and second binding compounds is a peptide.
 38. Theconjugate of claim 1, wherein the binding region of the first bindingcompound, the second binding compound, or each of the first and secondbinding compounds is independently selected from (CH₂)_(d)—CO₂ ⁻,—(CH₂)_(d)—SO₃ ⁻, —(CH₂)_(d)—OSO₃ ⁻, —(CH₂)_(d)—PO₃ ^(═, —(CH)₂)_(d)—OPO₃ ^(═), and —Ar—SO₃ ⁻.
 39. The conjugate of claim 1, whereinthe binding region of the first binding compound, the second bindingcompound, or each of the first and second binding compounds is—(CH₂)_(d)—CO₂ ⁻.
 40. The conjugate of claim 1, wherein the bindingregion of the first binding compound, the second binding compound, oreach of the first and second binding compounds is —(CH₂)_(d)—OSO₃ ⁻. 41.The conjugate of claim 1, wherein the binding region of the firstbinding compound, the second binding compound, or each of the first andsecond binding compounds is —(CH₂)_(d)—OPO₃ ^(═).
 42. The conjugate ofclaim 1, wherein the linker of the first binding compound, the secondbinding compound, or each of the first and second binding compounds is apolyvinylpyrrolidone.
 43. The conjugate of claim 1, wherein the linkerof the first binding compound, the second binding compound, or each ofthe first and second binding compounds is a polyethyleneglycol.
 44. Theconjugate of claim 43, wherein the polyethyleneglycol has a molecularweight in the range of 1,000 to 4,000.
 45. The conjugate of claim 43,wherein the linker of the first binding compound, the second bindingcompound, or each of the first and second binding compounds ispoly(ethyleneglycol)_(p), wherein p is from 40 to
 150. 46. The conjugateof claim 1, wherein the linker of the first binding compound, the secondbinding compound, or each of the first and second binding compounds ispolyglycolate.
 47. The conjugate of claim 1, wherein the linker of thefirst binding compound, the second binding compound, or each of thefirst and second binding compounds is polylactate.
 48. The conjugate ofclaim 1, wherein the linker of the first binding compound, the secondbinding compound, or each of the first and second binding compounds is acopolymer of polyglycolate or a copolymer of polylactate or a copolymerof polyglycolate and polylactate.
 49. The conjugate of claim 1, whereinthe first binding compound, the second binding compound, or each of thefirst and second binding compounds has a dissociation constant (K_(d))value equal to or greater than about 100 nM.
 50. The conjugate of claim1, wherein the first binding compound, the second binding compound, oreach of the first and second binding compounds has a dissociationconstant (K_(d)) value of at least about 100 nM.
 51. The conjugate ofclaim 1, further comprising: an effector molecule attached to thelamellar structure.
 52. The conjugate of claim 1, wherein the lamellarstructure has a void defined therein, and the conjugate furthercomprises an effector molecule contained within the void of the lamellarstructure.
 53. The conjugate of claim 1, further comprising: an effectormolecule incorporated within the lamellar structure.
 54. The conjugateof any one of claims 51-53, wherein the effector molecule is a cytotoxicagent.
 55. The conjugate of any one of claims 51-53, wherein theeffector molecule is a cytotoxic agent selected from fluorouracil andfluorouridine.
 56. The conjugate of any one of claims 51-53, wherein theeffector molecule is a paramagnetic agent.
 57. The conjugate of any oneof claims 51-53, wherein the effector molecule is a paramagnetic agentselected from Gd-DTPA, Gd-DOTA, Gd-DTPA-bis(methoxyethyl)amide, andMn-EDTA.
 58. The conjugate of any one of claims 51-53, wherein theeffector molecule is a sulfa antibiotic.
 59. The conjugate of any one ofclaims 51-53, wherein the effector molecule is a sulfa antibioticselected from sulfisoxazole, N′-(w-thiazolyl)sulfanilamide,sulfmethoxazole, and sulfisomidine.
 60. The conjugate of any one ofclaims 51-53, wherein the effector molecule is a radiopharmaceuticalagent.
 61. The conjugate of any one of claims 51-53, wherein theeffector molecule is a radioactive molecule emitting alpha, beta, orgamma radiation.
 62. The conjugate of any one of claims 51-53, whereinthe effector molecule is a radiopharmaceutical agent that comprises achelate of a beta emitting isotope or a chelate of a gamma emittingisotope.
 63. The conjugate of any one of claims 51-53, wherein theeffector molecule is a radiopharmaceutical agent that comprises Lu, Sm,In, Ru, or Tc.
 64. The conjugate of any one of claims 51-53, wherein theeffector molecule comprises a radionuclide selected from I-123, I-131,Tc-99m, Re-186, Re-188, Sm-152, Ho-155, Bi-202, and Lu-177.
 65. Theconjugate of any one of claims 51-53, wherein the effector molecule is aradiopharmaceutical agent selected from diethylenetriamine pentaaceticacid (DTPA) or 1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetraaceticacid (DOTA).
 66. The conjugate of any one of claims 51-53, wherein theeffector molecule is a chemotherapeutic agent.
 67. The conjugate of anyone of claims 51-53, wherein the effector molecule is selected fromhyaluronic acid and heparin.
 68. The conjugate of any one of claims51-53, wherein the effector molecule is an X-ray opacification agent.69. The conjugate of any one of claims 51-53, wherein the effectormolecule is an echogenic agent.
 70. The conjugate of claim 1, whereinthe lamellar structure comprises a micelle.
 71. The conjugate of claim1, wherein the lamellar structure comprises a liposome.
 72. Theconjugate of claim 1, wherein the lamellar structure comprises amicroparticle.
 73. The conjugate of claim 1, wherein the lamellarstructure comprises a nanoparticle.
 74. The conjugate of any one ofclaims 1-41, 49-53, and 70-73, wherein the linker of the first bindingcompound, the second binding compound, or each of the first and secondbinding compounds is a polynucleotide.
 75. The conjugate of any one ofclaims 1-41, 49-53, and 70-73, wherein the linker of the first bindingcompound, the second binding compound, or each of the first and secondbinding compounds is a copolymer of a polynucleotide.